The goal of this proposal is to demonstrate that a Guanine-rich (G-rich) segment in the promoter region of Tyrosine Hydroxylase (TH) can form multiple intramolecular G-Quadruplexes, with varying stability and conformation, and that these G-quadruplexes (GQs) differentially regulate TH expression at the transcription level. Single intramolecular GQ formed by four neighboring G-stretches located in promoter regions are known to regulate transcription. A critical but barely answered question is how ensemble of such GQs controls gene expression. To address this we will study a 45 nt segment of human TH promoter that contains seven stretches of Gs and is located within a conserved region, 24 nt upstream of the transcription start site. It is hypothesized that this G-rich segment can adopt multiple GQ structures that vary in stability and conformation and is a key regulatory element in TH promoter. Preliminary studies on the 45 nt DNA and a subset of its fragments suggest the presence of multiple GQs that vary in stability and conformation. This proposal aims to characterize the dynamicity and variability in conformations associated with the ensemble of GQs formed in the 45 nt segment by utilizing the unique capabilities of single molecule FRET and bulk fluorescence measurements, e.g. 2-aminopurine substitution, in combination with other biophysical and biochemical techniques. Furthermore, the stability and physiological relevance of these structures are tested against unfolding activity of human Replication Protein A (RPA), the most abundant single strand DNA binding protein in eukaryotes. In addition, the functionality of the various components of the 45 nt DNA in the context of the entire TH promoter will be determined by using GFP and luciferase reporter constructs using point mutation and deletion analysis. Understanding the underlying mechanism of TH gene expression is important because aberrant TH expression is linked to many psychiatric problems including bipolar disorder, manic depression, and schizophrenia. Additionally, loss of TH expressing neurons is a major factor in Parkinson's disease. The proposed studies will establish the structure-function relationship of the 45 nt segment in the context of regulation of TH promoter activity. Deciphering the molecular mechanism of TH promoter control will not only lead to better understanding of regulation of TH gene expression but may also identify new targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Tyrosine Hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis in the dopaminergic neurons and other tissues. Aberrant expression of Tyrosine Hydroxylase gene is linked to many psychiatric problems including bipolar disorder, manic depression, schizophrenia, and its deficiency is also associated with a broad spectrum of phenotype ranging from TH-deficient dopa-responsive dystonia (DRD) at the mild end to a levodopa-unresponsive infantile parkinsonism or progressive infantile encephalopathy phenotype at the severe end. Understanding of the molecular mechanism of TH promoter control will not only lead to better understanding of regulation of TH gene expression but may also identify new targets for therapeutic intervention of a wide variety of neurological disorders benefitting a wide spectrum of US population.